Trypanosomes use polycistronic transcription akin to the bacterial paradigm, but lacking the functional association between genes linked by transcription units. As such, transcriptional control of gene expression is minimal, which is of interest as several species are parasites responsible for human disease. Individual mRNAs are resolved into typical eukaryotic monocistronic mRNAs via 3' polyadenylation and 5' trans-splicing, the result of which is the presence of a universal exon sequence containing a hypermethylated cap 4 structure. The cap 4 is unique to the parasite, and is provided by the spliced leader RNA transcript, a small RNA that may travel through the cytosol in the course of its maturation prior to splicing. Trypanosomes possess six proteins related to the eukaryotic cap-binding translation-initiation factor eIF4E, as compared to three in humans. This family has the elements required to play major roles in gene expression for both spliced leader RNA processing and mRNA translation that are unique to the parasite, and hence provide a potential avenue for targeted clinical attack. Here we address the role and function of TbeIF4E5, which binds to three variants of the trypanosome translation initiation factor eIF4G and is otherwise uncharacterized. The goal of this application is to explore trypanosome-specific mechanisms of gene control mediated by their unique mRNA cap structure. The TbeIF4E family is our launching pad, with six homologues for eIF4E binding protein eIF4G and two for eIF4A adding to the combinatorial possibilities that may provide the finesse for modulation of gene expression. We begin with validation and expansion of our yeast two-hybrid protein-protein interaction results; an in vitro system will confirm these conclusions, followed by competition assays to determine association preferences. Functional redundancy of the eIF4E5 and eIF4E6 orthologs will be tested in gene knockdown and knockouts in Trypanosoma brucei with the goal of determining the function of eIF4E5. The proteins associating with eIF4E5 in vivo will be identified by purification of eIF4E5 complex followed by sensitive mass spectrometry; bound RNA and their cap structures will be examined by primer extension. This application is the basis of a collaboration between the Campbell/Sturm laboratory at UCLA and the laboratory of Dr. Osvaldo P de Melo Neto at the Research Center Aggeu Magalhaes (Brazil). Our groups approach these questions from opposing directions, with UCLA historically focused on RNA processing events leading up to trans-splicing and the Brazil work coming from the angle of translation. The budget includes the internship of a student from Brazil to UCLA, with a laboratory visit and seminar presented in Brazil. The UCLA group has an NIH award (AI056034; 7/01/09-6/30/14) to study the maturation of the kinetoplastid spliced leader RNA. The goals of this FIRCA application are not related directly to the parent grant. PUBLIC HEALTH RELEVANCE: The Order Kinetoplastida contains several pathogens of medical importance including the causative agents of human leishmaniasis, African trypanosomiasis and Chagas disease. Rather than using transcriptional regulation of gene expression, these pathogens employ unusual post-transcriptional mechanisms of regulation. Kinetoplastids possess multiple mRNA cap binding proteins, many of which do not have counterparts in the their mammalian hosts. The goal of this study is to identify unique components associated with the parasite-specific TbeIF4E5 protein, and its role in trypanosome biology.